Dietary cholesterol absorption and biliary cholesterol re-absorption contribute to plasma cholesterol homeostasis. Although individual variations in susceptibility to diet-induced hypercholesterolemia suggested a possible genetic influence on cholesterol absorption, the gene(s) involved with this process remains speculative. Previously published data and our preliminary results suggest that the pancreatic cholesterol esterase (CEL) plays a major role in cholesterol absorption. This research will use transgenic mice to test the hypothesis that differences in CEL gene expression contribute to individual variations in cholesterol absorption efficiency. The mechanism by which CEL catalyzes cholesterol absorption will also be explored. Additional experiments will be performed to test the hypothesis that CEL is necessary and sufficient to catalyze luminal cholesterol absorption under basal dietary conditions, but it acts in concert with other protein(s) for maximal cholesterol absorption in animals fed a high fat/high cholesterol diet. In Specific Aim 1, chimeric mice with targeted disruption of the CEL gene will be used to produce heterozygous and homozygous CEL-deficient mice to test the consequences of CEL-deficiency in lipid metabolism. Cholesterol absorption efficiency, plasma cholesterol level, hepatic LDL receptor, and HMG-CoA reductase activity will be compared in CEL(+/+), CEL(+/-), and CEL(-/-) mice fed a basal or a high fat/high cholesterol diet. Specific Aim 2 will produce transgenic mice with constitutively high luminal CEL,to test the effects of elevated CEL on cholesterol absorption and plasma cholesterol homeostasis. Specific Aim 3 will express mutagenized CEL in mice with a CEL(-/-) genetic background to determine if CEL-mediated cholesterol absorption is dependent on its catalytic activity and/or its ability to serve as a cholesterol carrier for lipid transport. A similar approach will be used in Specific Aim 4 to determine if CEL-mediated cholesterol absorption requires its interaction with heparin-like molecules on intestinal cells. Specific Aim 5 will evaluate cholesterol absorption efficiency and CEL mRNA level in selected inbred and recombinant inbred (RI) mice fed either basal or high fat/high cholesterol diet. The requirement of other protein(s), in addition to CEL, in catalyzing cholesterol absorption will be evaluated by segregational analysis of cholesterol absorption efficiency and CEL mRNA level among the RI mice strains. Results from these studies will help future design of intervention strategies aimed at lowering cholesterol absorption and reducing atherosclerosis in at-risk subjects.